Borislav Angelov

Multicompartment lipid cubic nanoparticles with high protein upload: millisecond dynamics of formation.

Membrane shapes, produced by dynamically assembled lipid/protein architectures, are crucial for both physiological functions and the design of therapeutic nanotechnologies. Here we investigate the dynamics of lipid membrane-neurotrophic BDNF protein complexes formation and ordering in nanoparticles, with the purpose of innovation in nanostructure-based neuroprotection and biomimetic nanoarchitectonics. The kinetic pathway of membrane states associated with rapidly occurring nonequilibrium self-assembled lipid/protein nanoarchitectures was determined by millisecond time-resolved small-angle X-ray scattering (SAXS) at high resolution. The neurotrophin binding and millisecond trafficking along the flexible membranes induced an unusual overlay of channel-network architectures including two coexisting cubic lattices epitaxially connected to lamellar membrane stacks. These time-resolved membrane processes, involving intercalation of discrete stiff proteins in continuous soft membranes, evidence stepwise curvature control mechanisms. The obtained three-phase liquid-crystalline nanoparticles of neurotrophic composition put forward important advancements in multicompartment soft-matter nanostructure design.

Small-Angle X-ray Scattering Investigations of Biomolecular Confinement, Loading, and Release from Liquid-Crystalline Nanochannel Assemblies

This Perspective explores the recent progress made by means of small-angle scattering methods in structural studies of phase transitions in amphiphilic liquid-crystalline systems with nanochannel architectures and outlines some future directions in the area of hierarchically organized and stimuli-responsive nanochanneled assemblies involving biomolecules. Time-resolved small-angle X-ray scattering investigations using synchrotron radiation enable monitoring of the structural dynamics, the modulation of the nanochannel hydration, as well as the key changes in the soft matter liquid-crystalline organization upon stimuli-induced phase transitions. They permit establishing of the inner nanostructure transformation kinetics and determination of the precise sizes of the hydrophobic membraneous compartments and the aqueous channel diameters in self-assembled network architectures. Time-resolved structural studies accelerate novel biomedical, pharmaceutical, and nanotechnology applications of nanochannel soft materials by providing better control of DNA, peptide and protein nanoconfinement, and release from diverse stimuli-responsive nanocarrier systems.

DNA/Fusogenic Lipid Nanocarrier Assembly: Millisecond Structural Dynamics

Structural changes occurring on a millisecond time scale during uptake of DNA by cationic lipid nanocarriers are monitored by time-resolved small-angle X-ray scattering (SAXS) coupled to a rapid-mixing stopped-flow technique. Nanoparticles (NPs) of nanochannel organization are formed by PEGylation, hydration, and dispersion of a lipid film of the fusogenic lipid monoolein in a mixture with positively charged (DOMA) and PEGylated (DOPE-PEG2000) amphiphiles and are characterized by the inner cubic structure of very large nanochannels favorable for DNA upload. Ultrafast structural dynamics of complexation and assembly of these cubosome particles with neurotrophic plasmid DNA (pDNA) is revealed thanks to the high brightness of the employed synchrotron X-ray beam. The rate constant of the pDNA/lipid NP complexation is estimated from dynamic roentgenograms recorded at 4 ms time resolution. pDNA upload into the vastly hydrated channels of the cubosome carriers leads to a fast nanoparticle-nanoparticle structural transition and lipoplex formation involving tightly packed pDNA.

Protein entrapment in PEGylated lipid nanoparticles

Defining appropriate delivery strategies of therapeutic proteins, based on lipid nanoparticulate carriers, requires knowledge of the nanoscale organization that determines the loading and release properties of the nanostructured particles. Nanoencapsulation of three cationic proteins (human brain-derived neurotrophic factor (BDNF), α-chymotrypsinogen A, and histone H3) was investigated using anionic nanoparticle (NP) carriers. PEGylated lipid NPs were prepared from self-assembled liquid crystalline phases involving monoolein and eicosapentaenoic acid. Inclusion of the antioxidant α-tocopherol favoured the preparation of stealth hexosome carriers. The purpose of the present work is to reveal the structural features of the protein-loaded lipid nanocarriers by means of high resolution small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). The obtained results indicate that protein entrapment is concentration-dependent and may significantly modify the inner liquid crystalline structure of the lipid nanocarriers through changes in the interfacial curvature and hydration.

Neurotrophin delivery using nanotechnology

Deficits or overexpression of neurotrophins cause neurodegenerative diseases and psychiatric disorders. These proteins are required for the maintenance of the function, plasticity and survival of neurons in the central (CNS) and peripheral nervous systems. Significant efforts have been devoted to developing therapeutic delivery systems that enable control of neurotrophin dosage in the brain. Here, we suggest that nanoparticulate carriers favoring targeted delivery in specific brain areas and minimizing biodistribution to the systemic circulation should be developed toward clinical benefits of neuroregeneration. We also provide examples of improved targeted neurotrophin delivery to localized areas in the CNS.

Long-living intermediates during a lamellar to a diamond-cubic lipid phase transition: a small-angle X-ray scattering investigation.

To generate nanostructured vehicles with tunable internal organization, the structural phase behavior of a self-assembled amphiphilic mixture involving poly(ethylene glycol) monooleate (MO-PEG) and glycerol monooleate (MO) is studied in excess aqueous medium by time-resolved small-angle X-ray scattering (SAXS) in the temperature range from 1 to 68 degrees C. The SAXS data indicate miscibility of the two components in lamellar and nonlamellar soft-matter nanostructures. The functionalization of the MO assemblies by a MO-PEG amphiphile, which has a flexible large hydrophilic moiety, appears to hinder the epitaxial growth of a double diamond (D) cubic lattice from the lamellar (L) bilayer structure during the thermal phase transition. The incorporated MO-PEG additive is found to facilitate the formation of structural intermediates. They exhibit greater characteristic spacings and large diffusive scattering in broad temperature and time intervals. Their features are compared with those of swollen long-living intermediates in MO/octylglucoside assemblies. A conclusion can be drawn that long-living intermediate states can be equilibrium stabilized in two- or multicomponent amphiphilic systems. Their role as cubic phase precursors is to smooth the structural distortions arising from curvature mismatch between flat and curved regions. The considered MO-PEG functionalized assemblies may be useful for preparation of sterically stabilized liquid-crystalline nanovehicles for confinement of therapeutic biomolecules.

Amphiphilic Branched Polymers as Antimicrobial Agents

Cationic amphiphilic polymers were prepared from PEI and functional ethylene carbonates bearing cationic, hydrophobic or amphiphilic groups. The polymers are designed to exhibit antimicrobial properties. In a one-step addition, different functional ethylene carbonates were added to react with the primary amine groups of PEI. The water soluble polymers were studied regarding their ability to form soluble aggregates. Their hydrodynamic radii, their inhibition potential against proliferation of E. coli and their hemolytic potential were determined. A structure-property relationship was established by analyzing the antimicrobial activity as a function of the ratio of alkyl to cationic groups, length of the alkyl chains, and molecular weight of the PEI.

Biocompatible Mesoporous and Soft Nanoarchitectures

Soft nanoarchitectures created by biomimetic self-assembly offer unexploited potential for therapeutic drug delivery applications, tissue engineering, and diagnostics. The lipid bilayer building blocks impart biocompatible properties and low toxicity of the resulting nanoassemblies. Our work provides a survey of the recent advances in design and structural studies of functional bicontinuous soft porous nanoarchitectures created from amphiphilic bilayer membrane building blocks. Depending on the packing symmetries and the densities of the curved lipid bilayers, organized in membrane-type nanoparticles, a class of multicompartment nanoobjects involving cubosomes, spongosomes, onion-like liposomes, or vesicles arranged in hierarchical supramolecular architectures, can be obtained. High resolution structural investigations by cryo-transmission electron microscopy microscopy and time-resolved small-angle X-ray scattering (SAXS) have demonstrated that binding and complexation of rigid protein molecules to flexible membrane–vesicle building blocks may generate significant changes in the curvature of the membrane interfaces and may induce formation of bicontinuous cubic nanoarchitectures. Enzyme-mediated cubic nanoarchitecture generation represents another low-energy fabrication method of nanoporous liquid crystalline assemblies. The kinetic pathway of packing ready-to-assemble membrane building blocks (vesicles, nanocubosomes) into nanoarchitectonic vehicles has been revealed by rapid-mixing stopped-flow SAXS experiments.

Earliest Stage of the Tetrahedral Nanochannel Formation in Cubosome Particles from Unilamellar Nanovesicles

Studies of nonequilibrium lipid polymorphism at the nanoscale contribute to the in-depth understanding of the structural pathways for formation of aqueous channels and emerging of channels-network ordering in liquid-crystalline (LC) nanovehicles. We present experimental structural evidence for the smallest tetrahedral-type lipid membrane aggregate, which involves completely formed nanochannels and occurs as an early intermediate state during the bilayer vesicle-to-cubosome particle transition. Nanovehicles are generated from a self-assembled lipid mixture and studied by means of high-resolution cryogenic transmission electron microscopy (cryo-TEM) and synchrotron radiation small-angle X-ray scattering (SAXS). The investigated lipid membrane composition allows for the stabilization of long-lived intermediates throughout the unilamellar vesicle-to-cubosome nanoparticle (NP) transformation at ambient temperature. The observed small cubosomic particles, with well-defined water channels, appear to be precursors of larger cubic membrane structures, thus confirming the theoretical modeling of nanochannel-network growth in diamond-type cubic lipid particles. The reported structural findings, highlighting that bilayer vesicle membrane packing and fusion are required for nanochanneled cubosome particle formation, are anticipated to advance the engineering of small lipid NPs with controllable channels for biomolecular loading and release.

Baicalin loaded in folate-PEG modified liposomes for enhanced stability and tumor targeting

Bioavailability of baicalin (BAI), an example of traditional Chinese medicine, has been modified by loading into liposome. Several liposome systems of different composition i.e., lipid/cholesterol (L), long-circulating stealth liposome (L-PEG) and folate receptor (FR)-targeted liposome (L-FA) have been used as the drug carrier for BAI. The obtained liposomes were around 80 nm in diameter with proper zeta potentials about -25 mV and sufficient physical stability in 3 months. The entrapment efficiency and loading efficiency of BAI in the liposomes were 41.0-46.4% and 8.8-10.0%, respectively. The morphology details of BAI lipsosome systems i.e., formation of small unilamellar vesicles, have been determined by cryogenic transmission electron microscopy (cryo-TEM) and small angle X-ray scattering (SAXS). In vitro cytotoxicity of BAI liposomes against HeLa cells was evaluated by MTT assay. BAI loaded FR-targeted liposomes showed higher cytotoxicity and cellular uptake compared with non-targeted liposomes. The results suggested that L-FA-BAI could enhance anti-tumor efficiency and should be an effective FR-targeted carrier system for BAI delivery.